Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR TREATING CHRONIC LYMPHOCYTIC
LEUKEMIA
FIELD OF INVENTION
The invention relates in general to treatment of chronic-lymphocytic leukemia
(CLL),
particularly to the treatment of aggressive CLL using HSP90 inhibitors; more
particularly to the
treatinent of CLL using ansamycins,. e.g., 17-allylamino-l7-
demethoxygeldanamycin (17-AAG).
BACKGROUND
The following description includes information that may be useful in
understanding the
present invention. It is not an admission that any of the information provided
herein is prior art
or relevant to the presently claimed inventions, or that any publication
specifically or implicitly
referenced is prior art.
The course of chronic lymphocytic leukemia (CLL) is variable. In aggressive
disease, CLL
cells usually express an unmutated immunoglobulin heavy-chain variable-region
gene (IgVH) and a
70-kD zeta-associated protein (ZAP70), whereas in indolent disease, the CLL
cells usually express
mutated IgVH but lack expression of ZAP70, The expression of ZAP70 in CLL
patients correlates
with disease progression, poor clinical outcome; decreased overall survival
and early requirement for
treatment. Although the presence of an unmutated IgVH is strongly associated
with the expression of
ZAP70, ZAP70 is a stronger predictor of the need for treatment and prognosis
in B-cell CLL.
Rassenti, L.Z. et al., NEngJMed., 2004, 351, 893-901.
ZAP70 is a 70-kD cytoplasmic protein tyrosine kinase (PTK) that ordinarily is
expressed
only in natural killer (NK) cells and T-cells and plays a critical role in =T-
cell-receptor signaling.
Keating et al. Hematology, 2003, 153-175. B-cells lack ZAP70, and instead use
another related PTK
for signal transduction via the B-cell receptor (BCR) complex. Studies, found
that CLL B cells that
have unmutated IgVH genes generally expressed levels of ZA.P70 protein that
were comparable to
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those expressed by normal blood T cells. In contrast, CLL B cells that
expressed mutated IgVH
genes, or that had low-level expression of CD3 8, generally do not express
detectable.levels of the
ZAP70 protein. Chen, et al. Blood 2002, 100:13, 4609-4614. B-cell expression
of ZAP70 is not
genetically predetermined. Chen, 2002, supra.. Expression of ZAP70 has
functional significance for
the signaling capacity of the BCR complex expressed in CLL: Keating et al.
supra. ZAP70
promotes phosphorylation of downstream signaling molecules after engagement of
the BCR and
plays a role in membrane antigen-receptor signaling 'pathways. Keating et al.
supra and Rassenti et
al. supra. One study shows that expression of ZAP70 in CLL allows for more
effective IgM-
signaling in CLL B cells, a feature that could contribute to the relatively
aggressive clinical behavior
generally associated with CLL cells that express unmutated IgVH. Chen, et al.
Blood, 2004,
prepublication online October 28, 2004.
The eukaryotic heat shock protein 90s (HSP90s) are ubiquitous chaperone
proteins involved
in folding, activation and assembly of a wide range of client proteins,
including mediators of signal
transduction, cell cycle control and transcriptional regulation. In order to
exert its function on client
proteins, HSP90 requires the formation of an active protein complex composed
of cochaperone
molecules and an active ATP binding site. Proteins identified as HSP90 client
proteins include
transmembrane tyrosine kinases [HER-2/neu, epidermal growth factor receptor
(EGFR), MET and
insulin-like growth factor-1 receptor (IGF-1R)], metastable signaling proteins
(Akt, Raf-1 and IKK),
mutated signaling proteins (p53, Kit, Flt3 and v-src), chimeric signaling
proteins (NPM-ALK, Bcr-
Abl), steroid receptors (androgen, 'estrogen and progesterone receptors), cell-
cycle regulators (cdk4,
cdk6) and apoptosis related proteins. It has been postulated that malignant
progression and cancer
prognosis may be associated with the presence of activated HSP90 which exists
in heightened complexes with cochaperone proteins. Kamal et al., Nature, 2003,
425:407-410.
Ansamycin antibiotics, e.g., herbimycin A (HA), gelda.namycin (GDM),17-AAG,
and other
HSP90 inhibitors are thought to exert their anticancerous effects by tight
binding of the N-terniinus
ATP-binding pocket of HSP90 (Stebbins, C. et al., Cell,1997, 89:239-250). This
pocket is highly
conserved and has weak homology to the ATP-binding site of DNA gyrase
(Stebbins, C. et al.,
supra; Grenert, J.P. et al., J. Biol. Chem. 1997, 272:23843-50). Further, ATP
and ADP'have both
been shown to bind this pocket with low affinity and to have weak ATPase
activity (Proromou, C. et
al., Cell;1997, 90: 65-75; Panaretou, B. et al., EMBO J.,1995,17: 482936). In
vitro and in vivo
studies have demonstrated that occupancy of this N-terminal pocket by
ansamycins and other HSP90
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inhibitors alters HSP90 function and inhibits protein folding. At high
concentrations, ansamycins
and other HSP90 inhibitors have been shown to prevent binding of protein
substrates to HSP90
(Scheibel, T., H. et al., Proc. Natl. Acad. Sci. USA, 1999, 96:1297-302;
Schulte, T. W. et al. J. Biol.
Chem. 1995, 270:24585-8; Whitesell, L. et al. Proc. Natl. Acad. Sci. USA,
1994, 91:8324-8328).
Ansainycins have also been demonstrated to inhibit the ATP-dependent release
of chaperone-
associated protein substrates (Schneider, C. L. et al., Proc. Natl. Acad. Sci.
USA, 1996, 93:14536-41;
Sepp-Lorenzino et al. J. Biol. Chem. 1995, 270:16580-16587). Izi either event,
the substrates are
degraded by a ubiquitin-dependent process in the proteasome (Schneider, C. L.
supra; Sepp-
Lorenzino, L. et al. J. Bi.ol. Chem., 1995, 270:16580-16587; Whitesell, L. et
al., supra).
This substrate destabilization occurs in both tumor and non-transformed cells
alike and has
been shown to be especially effective on a subset of signaling regulators,
e.g., Raf (Schulte, T. W. et
al., Biochem. Biophys. Res. Commun. 1997, 239:655-9; Schulte, T. W. et al. J.
Biol. Chem. 1995, =
270:24585-8), nuclear steroid.receptors (Segnitz, B., and U. Gehring, J. Biol.
Chem. 1997;
272:18694-18701; Smith, D. F. et al. Mol. Cell. Biol. 1995, 15:6804-12 ), v-
src (Whitesell, L., et al.,
Proc. Natl. Acad. Sci. USA, 1994, 91:8324-8328) and certain transmembrane
tyrosine kinases (Sepp-
Lorenzino, L. et al. J. Biol. Chem. 1995, 270:16580-16587) such as EGF
receptor (EGFR),
Her2/Neu (Hartmann, F. et al. Int. J. Cancer 1997,.70:221-9; Miller, P. et
al., Cancer Res. 1994,
54:2724-2730; Mimnaugh, E. G. et al. J. Biol. Chem. 1996, 271:22796-801;
Schnur, R. et al., J.
Med. Chem. 1995, 38:3806-3812), CDK4, and mutant p53 (Erlichman et al., Proc.
AACR, 2001, 42,
abstract 4474). The ansamycin-induced loss of these proteins leads to the
selective disruption of
certain regulatory pathways and results in growth arrest at specific phases of
the cell cycle (Muise-
Heimericks, R. C. et al.; J. Biol. Chem., 1998, 273:29864-72), and apoptosis,
and/or differentiation
of cells so treated (Vasilevskaya, A. et al., Cancer Res., 1999, 59:3935-40).
Because ZAP70expression is associated with an aggressive form of CLL, a means
of
controlling such an overexpression is needed. A treatment that could
simultaneously avoid or
minimize harm to normal cells and tissues would be most desirable. The present
invention addresses
these needs..
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SUMMARY OF THE INVENTION
The inventors of the present invention have found that ZAP70 is a client
protein of
HSP90 and that specific inhibitors of HSP90, such as 17-AAG, down modulate the
expression
and function of this tyrosine kinase and induce apoptosis preferentially in
ZAP-90 positive.CLL
B cells in a dose- and,time-dependent manner.
One aspect of the invention is a method of treating a form of CLL which is
characterized
by the expression of ZAP70 in the CLL B cells by administering to a patient in
need thereof a
pharmaceutically effective amount of a HSP90 inhibitor.
In one embodiment, the inhibitor is.an ansamycin.;. and the ansamycin is
selected from the
group below, or a polymorph, solvate, ester, tautomer, enantiomer,
pharmaceutically acceptable
salt or prodrug thereof:
/~O
O H. 0 ZOH 0
O N O
H 0 H ~/
OH a OH ~O O
O 0 O 0 0
a
HZN~o H2N~0 HZN~O
Geldanamycin DMAG 17-AAG
OY H 00 H H H 0
N~ O 50 ONI 1 1 1NO
H / 0 H 0 AOHHINHzHzN O
Compound #563 Compound #237
CN I O
O O H O
ON 1 1
0 H H
OH 0 O OH ~O
0 0 O O
-~0
HOH2N-~-10 H2N0
Compound #956 Compound # 1236
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In one further embodiment, the ansamycin is 17-AAG which may comprise low melt
'forms of 17-AAG characterized by DSC melting temperatures below 175 C and/or
by an X-ray
powder diffraction pattern having peaks located at 5.85 degree, 4.35 degree
and 7.90 degree two-
theta angles. In another embodiment, the ansamycin is a low melt polymorph of
17-AAG which
is characterized by a DSC melting temperature at about 156 C and by an X-ray
powder
diffraction pattern having peaks located at 5.85 degree, 4.35 degree and 7.90
degree two-theta
angles. In yet another embodiment, the ansamycin is another low melt polymorph
of 17-AAG
characterized by a DSC melting temperature at about 172 C. Further, the 17-
AAG may be a
high melt form, a low melt form, an amorphorus form, or combination thereof.
In yet other embodiment; the inhibitor binds at the ATP-binding site of a
HSP90.
In another aspect of the invention, the HSP90 inhibitor is administered
intravenously,
intralesionally, parenterally, or orally. -
In a further aspect of the invention, the HSP90 inhibitor has an IC50 between
about two to
10 fold lower for the HSP90 in the B cells of the patient having elevated
ZAP70 than the HSP90
in the normal B cells that do not have elevated ZAP70. In one embodiment, the
HSP90 inhibitor
has an ICSO about two fold, 5 fold or 10 fold lower for the HSP90 in the B
cells of the patient
having elevated ZAP70 than the HSP90 in the normal B cells that do not have
elevated ZAP70.
In another aspect of the invention, the inhibitor exhibits an IC50 of about
100 nM or less
in the cells having elevated ZAP70. In one embodiment, the inhibitor exhibits
an IC50 of about
75 nM or less in the cells having elevated ZA.P70. In one embodiment, the
inhibitor exhibits an
ICso of about 50 nM or less in the cells having elevated ZAP-70. In a fiu-ther
embodiment, the
inhibitor exhibits an IC50 of about 30 nM in the cells having elevated ZAP70.
The above aspects and embodiments may be combined when feasible or
appropriate.
Other aspects and variation of the forgoing aspects and embodiments which are
obvious to those
skilled in the art are within the contemplation of the invention.
Advantages of the invention include one or more of ease of manufacture, the
use of
clinically acceptable reagents (e.g., having reduced environment and/or
patient toxicity),
enhanced formulation stability, less complicated shipping and warehousing, and
simplified
pharmacy and bed-side handling. Other advantages, aspects, and embodiments
will be apparent
from the description above and the detailed description and claims to follow.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows the competitive binding of 17-AAG against a biotinylated
geldanamycin probe (biotin-GM) for HSP90 in lysates of B cells, T cells, ZAP70
positive CLL B
cells (ZAP70+ CLL B cells) and ZAP70 negative CLL B cells (ZAP70- CLL B
cells). The
Western blot bands show that inhibition of binding of HSP90 to the biotin-GM
decreases with
increasing concentration of 17-AAG (l a.). The results are quantitated and
plotted in %
inhibition of binding of HSP90 to the biotin-GM vs. 17-AAG concentration in nM
(1b). The
IC50 reported is the concentration of 17-AAG needed to cause half-maximal
inhibition of binding
(1c).
FIGURE 2 presents graphically the inhibition of binding of biotinylated
geldanamycin
probe (biotin-GM) for HSP90 in lysates of ZAP70+ CLL B cells (A) and ZAP70-
CLL B cells
(M) in the presence of increasing concentrations of 17-AAG .
FIGURE 3 presents graphically the inhibition of binding of biotinylated
geldanamycin
(biotin-GM) for HSP90 in lysates of normal B cells (+) and T cells 15 FIGURE 4
demonstrates the association of HSP90 and ZAP70 in MCF-7 breast
carcinoma cells, ZAP70+ CLL B and ZAP70- CLL B and normal T and B cells by co-
inununoprecipation and analyzed by SDS-PAGE and Western blots using the
indicated
antibodies. "IP" denotes immunoprecipation and "WB" denotes Western Blot. P23
and HOP
are essential components of two known multi-chaperone HSP90 complexes.
FIGURE 5 compares the degradation of ZAP70 in ZAP70+ CCL B cell after
treatment
with EC1 (17-AAG) (M), EC82 (A), EC86 (X) (EC82 and EC86 are purine based
HSP90
inhibitors) or EC 116 (an inactive structurally-related HSP90 inhibitor) (+)
for 24 hours at 37 C.
FIGURE 6 compares by two-color flow cytometry the expression of ZAP70 in CLL B
cells untreated (left pan,el) or treated with 300nM EC1 (17-AAG) (right panel)
for 24 hours at
37 C. The upper right quadrrant were normal T-cells (CD3+, ZAP70+) the lower
right quadrant
were (CD3-, ZAP70+); the upper left quadrant is CD3+, ZAP70-; and the lower
left quandrant is
CD3-, ZAP70-.
FIGURE 7 compares the % viability (expressed as 100% - % apoptotic cells) of
ZAP70+
CCL B cells after treatment with EC1 (17-AAG) (~) or EC116 (inactive
structurally-related
HSP90 inhibitor)
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FIGURE 8 compares the % viability (expressed as 100% - % apoptotic cells) of
ZAP70+
CCL B cells after treatment with 100 nM of EC1 (17-AAG) (0) or ECI16 (inactive
structurally-
related HSP90 inhibitor) (*)The time taken to reach 50% cell mortality is
approximately 48
hours after treating with 17-AAG.
FIGURE 9 compares the viability (expressed as 100% - % apoptotic cells) of CCL
B
cells) from sixteen ZAP70+ patients and eleven ZAP70- patients after treatment
with 100 nM
EC1 (17-AAG) for 48 hours. ZAP70+ CLL B cells have an average % viability of
45.74 +/-
3.177%, whereas ZAP70- CLL B cells have an average % viability of 93 +/-1.701
%. The
Students T-Test P-value of the difference in survival between the two
populations was < 0.0001.
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to methods of treating an aggressive form of chronic
lymphocytic leukemia (CLL) which is characterized by over expression of ZAP70,
a protein
kinase which normally found only in T cells, with HSP90 inhibitors. The method
is based on the
observation that ZAP70 co-immunoprecipates with HSP90, suggesting that it is
anHSP90 client
protein. The inventors further observed that in ZAP70 positive CLL samples,
the majority of
HSP90 present in the cytoplasm was in a complexed form, whereas in ZAP70
negative samples,
most HSP90 was found to be uncomplexed. The inventors hypothesized that the
abnormal
overexpression and function in CLL B cells may depend on activated HSP90 and
its level of
expression may be down-regulated by using a specific HSP90 inhibitor, leading
to induction of
apoptosis of ZAP70 positive CLL B cells. It was found that the level of ZAP70
expression was
decreased 30-40% in cells treated for 24 hours with HSP90 inhibitors at
nanomolar
concentrations.
1. DEFINITIONS
The following terms have the following meanings and terms not specifically
appearing
below have their common customary meaning as used in the art:
The term "ZAP70 positive" and "ZAP70 negative" are based on a cutoff
expression of
20% as measured by flow cytometry (FACSCalibur, BD Biosciences and Flow 7o
software).
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An "HSP90-inhibiting compound" or "HSP90-inhibitor" is one that disrupts the
structure
and/or function of an HSP90 chaperone protein and/or a protein that is
dependent on HSP90.
HSP90 proteins are highly conserved in nature (see, e.g., NCBI accession #'s
P07900 =and XM
004515 (human (x and (3 HSP90, respectively), P11499 (mouse), AAB2369 (rat),
P46633
(chinese hamster), JC 1468 (chickeri), AAF69019 (flesh fly), AAC21566
(zebrafish), AAD3 0275
(salmon), 002075 (pig), NP 015084 (yeast), and CAC29071 (frog)). Grp94 and
Trap-1 are
related molecules falling within the definition of an HSP90 as used herein:
There are thus many
different HSP90s, all with anticipated similar effect and inhibition
capabilities. The HSP90
inhibitors of the invention may be specifically directed against an HSP90 of
the specffic host
patient or may be identified based on reactivity against an HSP90 homolog from
a different
species or an HSP90 variant.
The term "ansamyciri" is a broad term which characterizes compounds having an
"ansa"
structure which comprises any one of benzoquinone, benzohydroquinone,
naphthoquinone or
naphthohydroquinone moities bridged by a long chain. Compounds of the
naphthoquinone or
naphthohydroquinone class are exemplified by the clinically important agents
rifampicin and
rifamycin, respectively. Compounds of the benzoquinone class are exemplified
by
geldanamycin (including its synthetic derivatives 17-allylamino-17-
demethoxygeldanamycin
(17-AAG), 17 N,N-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG),
dihydrogeldanamycin and herbamycin). The benzohydroquinone class is
exemplified by
macbecin. While the invention is illustrated using ansamycins, in particular,
17-AAG, it should
be understood that the novel method of treating CLL described herein applies
to both the high
melt and low melt forms of the compound, and its polymorphs, tautomeirs,
enantiomers,
pharmaceuticaIly acceptable salts, and.prodrugs. It should be further
understand that the method
further applies to many other ansamycins including, but not limited to, those
exemplified in
Examples 1-13 of the EXAMPLE section, such as geldanamycin, 17-N,N-
dimethylaminoethylarninogeldanamycin, and polymorphs, tautomers, enantiomers,
pharmaceutically acceptable salts, and prodiugs thereof. The structures of the
numbered
compounds are disclosed in the Summary section.
The term "pharmacologically active compound," "active pharmaceutical
ingredient" or
"therapeutical ingredient" is synonymous with "drug" and means any compound
that exerts,
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directly or indirectly, a biological effect, in vitro or in vivo when
administered to cultured cells or
to an organism.
A "prodrug" is a drug covalently.bonded to a carrier wherein release of the
drug occurs in
vivo when the prodrug is administered to a mammalian subject. Prodrugs of the
compounds of
the present invention are prepared by modifying functional groups present in
the compounds in
such a way that the modifications are cleaved, either in routine manipulation
or in vivo, to yield
the desired compound. Prodrugs include compounds wherein hydroxy, amine, or
sulfhydryl
groups are bonded to any group that; when administered to a mammalian subject,
is cleaved to
form a free hydroxyl, amino, or sulfhydryl group, respectively. Examples of
prodrugs include,
but are not limited to, acetate, formate, or benzoate derivatives of.alcohol
or amine.,functional
groups in the compounds of the present invention; phosphate esters;
dimethylglycine esters,
arninoalkylbenzyl esters, amirioalkyl esters or carboxyalkyl esters of alcohol
or phenol functional
groups in the compounds of the present invention; or the like. Prodrugs can
impart multiple
advantages for drug delivery, e.g., as explained in REMINGTON PHARMACEUTICAL
SCIENCES, 20th
Edition, Ch. 47, pp. 913-914.
"Pharmaceutically acceptable salts" include those derived from
pharmaceutically
acceptable inorganic and organ.ic acids and bases. Examples of suitable acids
include
hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic,
phosphoric, glycolic,
gluconic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic,
citric, methanesulfonic,
formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, 1,2
ethanesulfonic acid
(edisylate), galactosyl-d-gluconic acid and the like. Other acids, such as
oxalic acid, while not
themselves pharmaceutically acceptable, may be employed in the preparation of
salts useful as
intermediates in obtaining the compounds of this invention and their
pharmaceutically acceptable
acid addition salts. Salts derived from appropriate bases include alkali metal
(e.g., sodium),
alkaline earth metal (e.g., magnesium), ammonium and N-(Cl-C~ alkyl) a+ salts,
and the like.
Illustrative examples of some of these include sodium hydroxide, potassium
hydroxide, choline
hydroxide, sodium carbonate, and the like. Where the claims recite "a compound
(e.g.,
compound 'x') or pharmaceutically acceptable salt thereof," and only the
compound is displayed,
those claims are to be interpreted as embracing, in the alternative or
conjunctive, a
pharmaceutically acceptable salt or salts of such compound.
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A"pharmaceutically effective amount" means an amount which is capable of
providing a
therapeutic or prophylactic effect. The specific dose of compound administered
according to this
invention to obtain therapeutic and/or prophylactic effect will, of course, be
determined by the,
particular circumstances surrounding the case, including, for example, the
specific compound
administered, the route of administration, the condition being treated, and
the individual being
treated. A typical daily dose (administered in single or divided doses) will
contain a dosage level
of from about 0.01 mg/kg to about 100 and more preferably 50 mg/kg of body
weight of an
active compound of this invention. Preferred daily doses generally will be
from about
0.05 mg/kg to about 20 mg/kg and ideally from about 0.1 mg/kg to about 10
mg/kg.
The preferred therapeutic effect is the inhibition, to some extent, of the
growth of cells
characteristic of the disorder. treated. A therapeutic effect will also
normally, but need not,
relieve to some extent one or more of the symptoms associated with the
disorder.
The term "IC50" is defmed as the concentration of an HSP90 inhibitor required
to achieve
killing of 50% of the cells of a population, or of a particular cell type,
e.g., cancerous versus
noncancerous cells within a greater cell population. The IC50 is preferably,
although not
necessarily, greater for normal cells than for cells exhibiting a
proliferative disorder.
A "physiologically acceptable carrier" refers to a carrier or diluent that
does not cause
significant irritation to an organism and does not abrogate the biological
activity and properties
of the administered coinpound. Depending on the formulation, the diluent can
be a solid such as
calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium
phosphate, or a
liquid, such as water or oils.
An "excipient" refers to a non-toxic pliarmaceutically acceptable substance
added to a
pharmacological composition to facilitate the processing, administration and
pharmaceutics
properties of a compound. Excipients may include but are not limited to,
fillers, diluents,
glidants, lubricants, disintegrants, binders, solubilizers,
stabilizers/bulking agents, and various
functional and non-functional coatings.
The term "about" means including and exceeding up to 15% the specific
endpoint(s)
designated. Thus the range is broadened.
The term "optionally" denotes that the step or component following tlie'term
may but 30 need not be a part of the method or formulation.
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II. PREPARATION OF ANSAMYCINS
Ansamycins according to this invention may be synthetic, naturally-occurring,
or a
combination of the two, i.e., "semi-synthetic," and may include dimers and
conjugated variant
and prodrug forms. Some exemplary benzoquinone ansamycins useful in the
various
embodiments of the invention and their methods of preparation include but are
not limited to
those described, e.g., in U.S. Patent No. 3,595,955 (describing the
preparation of geldanamycin),
No. 4,261,989, No. 5,387,584, and No. 5,932,566 and those described in the
"EXAMPLE"
section (Examples 1-12), below. Geldanamycin is also commercially available,
e.g., from CN
Biosciences, an Affiliate of Merck KGaA, Darmstadt, Germany, headquartered in
San Diego,
California, USA (cat. rio. 345805). 17-N,N-dimethylaminoethylamino-17-
desmethoxy-
geldanamycin (DMAG) is commercially available from EMD/Calbiochem. The
biochemical
purification of the geldanamycin derivative, 4,5-dihydrogeldanamycin and it's
hydroquinone
from cultures of Streptomyces hygroscopicus (ATCC 55256) are described in WO
93/14215
(Cullen et al.): An alternative method of synthesis for 4,5-
dihydrogeldanamycin by catalytic
hydrogenation of geldanamycin is also known. See e.g., "Progress in the
Chemistry of Organic
Natural Products," Chemistry of the Ansamycin Antibiotics, 1976 33:278. Other
ansamycins that
can be used in connection with various embodiments of the invention are
described in the
literature cited in the "Background" section and also inthe "Summary" section,
above.
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17-AAG may be prepared from geldanamycin by reacting with allyamine in dry THF
under a nitrogen atmosphere. The crude product may be purified by slurrying in
H20:EtOH
(90:10), and the washed crystals obtained have a melting point of 206-212 C
by capillary
melting point technique. A second product of 17-AAG can be obtained by
dissolving and
recrystallizing the crude product from 2-propyl alcohol (isopropanol). This
second 17-AAG
producthas a melting point between 147-153 C by capillary melting point
technique. The two
17-AAG products are designated as the low melt form and high melt form. The
stability of the
low melt form maybe tested by slurring the crystals in the solvent (H20:EtOH
(90:10)) from
which the high melt form was purified; 'no conversion to the high melt form
was observed. See
Examples 1-2 for. details of the preparation of the two polymorphic forms of
17-AAG.
III. CHARACTERIZATION AND EVALUATION OF THE EFFECTIVENESS OF DowN REGULATING
ZAP70 BY INHIBITION OF HSP90
A. Determining ZAP70 Levelsin Cell Lysates
Many different types of methods are known in the art for determining protein
concentrations and measuring or predicting the level of proteins within cells
and in fluid
samples. Indirect techniques include nucleic acid hybridization and
amplification using, e.g.,
polymerase chain reaction (PCR). These techniques are known to the person of
skill and are
discussed, e.g., in Sambrook, Fritsch & Maniatis, MOLECULAR CLONING: A
LABORATORY
MANUAI, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
N.Y., Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John'Wiley &
Sons, NY,
1994. The concentration of ZAP70 may=be determined by immunoassay techniques
such as
immunoblotting, radioimmunoassay, immunofluorescence, western blotting,
immunoprecipitation, enzyme-linked immunosorbant assays (ELISA), and.
derivative techniques
that make use of antibodies directed against ZAP70, and flow cytometry. A
convenient and
quantitative method of deterxnining ZAP70 expression is FACS (fluorescence-
activated cell
soirter) (FACSCalibur, BD Biosciences and Flow-Jo software, version 2.7.4
(Tree Star)), a
version of flow cytometry, which is described in a recently published study by
Rassenti et al.
supra and the disclosure of which is incorporated herein by reference. In the
Rassenti study, the
blood cells were stained with CD19-specific and CD3-specific monoclonal
antibodies conjugated
with allophycocyanin and phycoerythrin, respectively (Pharmingen) and also
with anti-ZAP70
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monoclonal antibody that had been conjugated to Alexa-488 dye (Becton
Dickenson). Other dye
systems may also be used. Lymphocytes were gated on the basis of their forward-
angle light
scatter and side-angle light scatter, and.blood mononuclear cells from a
healthy donor can be
used to establish the initial gate. The expression of ZAP70 was meastired by
calculating the
percentage of CD19+CD3- cells that was above this gating threshold. ZAP70
positi've and
ZAP70 negative can be based on a cutoff expression, e.g:, as expression of
ZAP70 detected by
flow cytometry in more than 20% of leukemia cells.
B. Determining the Binding Affinity of HSP901igands to HSP90
A variety of isotopic and nonisotopic methods, e.g., colorimetric, enzymatic,
and
densitometric, afford sufficient sensitivity to evaluate the binding affinity
of an inhibitor to a
target protein. These methods are generally known in the art and can be used
in the context of
this invention. -
The binding affinity of HSP901igands to. HSP90 can also be measured by the
competitive
binding assay described in Kamal et al., Nature 2003, 425:407-410, the
disclosure of which is
incorporated herein by reference. The binding affinity of the ligand is
measured' by its ability to
inhibit the binding of geldanamycin, a known inhibitor of HSP90. The cell
containing the
HSP90 is first lysed in lysis buffer. The lysates were incubated with or
without 17-AAG and
then incubated with biotin-GM linked to BioMagTM streptavidin magnetic beads
(Qiagen). The
bound samples and the unbound supernatant can be separately collected and
analyzed on SDS
protein gels, and blotted using an HSP90 antibody (StressGen, SPA-830). The
bands in the
Western blots may be quantitated using the Bio-rad Fluor-S MultiImager, and
the % inhibition of
binding of HSP90 to the biotin-GM was calculated. The IC50 is the
concentration of HSP90
ligand needed to cause half-maximal inhibition of binding.
FIGURES 1-3 show the competitive binding of 17-AAG against a biotinylated
geldanamycin probe (biotin-GM) for HSP90 in lysates of B cells, T cells, ZAP70
positive CLL B
cells and ZAP70 negative CLL B cells. The Western blot bands show that
inhibition of binding
of HSP90 to the biotin-GM -decreases with increasing concentration of 17-AAG
(1 a.). The
results are quantitated and plotted in % inhibition of binding of HSP90 to the
biotin-GM vs. 17-
AAG concentration in nM (lb). The figures show that the inhibition of binding
is higher for
HSP90 isolated from ZAP70+ CLL B cells. The calculated IC50 shows that 17 AAG
has an
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approximately I Ox higher binding affinity for HSP90 isolated from ZAP70+ CLL
B cells
compared to ZAP70- CLL B cells and to normal B cells.
C. Determining the Association of the Proteins by Co-immunoprecipitation
The inter-association of various proteins can be determined by co-
immunoprecipation
experiments using antibodies specific for the proteins of interest. These
methods are well known
in the art. ' See, Goldsby R.A. et al., KTRBY IMMUNOLOGY, 4th Edition, W.H.
Freeman and
Company, 2000.
To determine whether ZAP70 is a client protein of HSP90 and whether HSP90 in
the
ZAP70+ CLL B cells is present in multi-chaperone complexes, a set of four co-
immunopreciptation experiments were performed. MCF-7 breast carcinoma oells,
primary
isolated of ZAP70+ and ZAP70- chronic lymphocytic leukemia (CLL B) cells and
normal T and
B cells were lysed and incubated with pre-blocked protein-A Sepharose beads
(Zymed) with
antibodies specific for the protein of interest. The bound and unbound
fractions can be
separately collected and analyzed by SDS-PAGE and Western blots using the
indicated
antibodies.
FIG. 4 shows the immunoblot of the co-imrnunoprecipitation experiment. The
antibodies
used in each step of the experiment were indicated. IP Ab denotes the antibody
used during
immunoprecipation. WB Ab denotes the antibody used during Western Blot. P23
and HOP are
essential components of the two known multi-chaperone HSP90 complexes. The
first gels
demonstrate that ZAP70 is expressed in ZAP70+ CLL B cells and normal T cells,
but not in
ZAP70- CLL B cells or normal B cells. The second gels show that ZAP70 is
physically
associated with HSP90 in ZAP70+ CLL B cells, but not in any of the other cell
types, including
normal T cells. The third gels confirm the previous finding by reversing the
co=
immunoprecipitation. The fourth gels show that HSP90 in MCF-7 cells (the
positive control, see
Kamal et al., Nature, 2003, 425:407-410) and in Zf1.P70-+ CLL B cells is in
activated state
(multichaperone complexes with HOP and p23), whereas HSP90 in ZAP70- CLL B
cells or
normal T or B cells is in the latent resting state (not associated with HOP or
p23).
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IV. CHARACTERIZATION AND EVALUATION OF THE EFFECTIVENESS OF INHIBITION OF
ZAP70
The downstream effect on ZAP70 by inhibition of HSP90 can be directly measured
by
the amount of ZAP70 expression or by determining the viability of the cells
after treatment with
selected HSP90 inhibitors.
Primary isolates of B-cell chronic lymphocytic leukemia cells from an
individual
ZAP70 patient were treated with EC 1(17-AAG), EC82 or EC86 (two purine based
known
HSP90 inhibitors) or EC 116 (an inactive structurally-related HSP90 inhibitor)
for 24 hours at
37 C. Levels of ZAP70 protein expression were measured by indirect
immunofluorescence of
permeabilized cells with specific anti-ZAP70 antibodies and FACS analysis.
FIGURE 5 shows that all three active HSP90 inhibitors dose-dependently induced
degradation of ZAP70, confirming that ZAP70 is an HSP90-dependent .client
protein, as was
'indicated by the physical association demoristrated in the co-
immunoprecipitation experiments
(FIG. 4). Additionally, the fact that three structurally-unrelated HSP90
inhibitors produced the
same effect strongly implicates HSP90 as an essential protein for the
stability of ZAP70 in CLL
B cells.
Primary isolates of white blood cells from an individual ZAP70+ B-cell chronic
lymphocytic leukemia patient were left untreated (left panel) or treated with
300 nM EC 1 (17-
AAG) for 24 hours at 37 C (right panel). Levels of ZAP70 protein expression
were measured by
two color indirect immunofluorescence with specific anti-CD3 antibodies
conjugated to
phycoerythrin and anti-ZAP70 antibodies conjugated to Alexa-488 dye and
analyzed by flow
cytometry. CD3 is a specific marker of T cells. FIGURE 6 compares the
expression of ZAP70
in untreated CLL-B cells untreated (left panel) or treated (300 nM 17-AAG)
(right panel) cells.
As shown in the untreated cells (left panel), approximately 5% of the cells
were normal T-cells
(CD3+, ZAP70+, upper right quadrant) and -85% of the cells were ZAP70+ CLL B
cells (CD3-,
ZAP70+, lower right quadrant). EC 1(17-AAG) induced degradation of ZAP70 in
the B-CLL
cells (% positive cells 85% -> 34%), but not in the nozmal T-cells (% positive
cells 4.5% ~
4.2%), as predicted from the physical association demonstrated in B-CLL cells,
but not in the
normal T-cells in the co-immunoprecipitation experiments (FIGURE 4). The
fmding that HSP90
inhibitors induce ZAP70 degradation in B-CLL cells but not normal T-cells
indicates that such
drugs would have a more specific antileukemic activity than ZAP70 kinase
inhibitors. This is
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important because B-CLL patients are chronically immuinosuppressed by their
disease, so
avoidance of effects on normal T-cell function performed by ZAP70 is clearly
beneficial.
Primary isolates of CLL B cells from an individual ZAP70+ patient were treated
with
EC1 (17-AAG) or EC116 (inactive structurally-related HSP90 inhibitor) for 48
hours at 37 C.
Apoptotic cells were identified by a standard protocol using the mitochondrial
vital dye DiOC6
and propidium iodide staining. The % viability is expressed as 100% - %
apoptotic cells.
FIGURE 7 shows compares the % viability of ZAP70+ chronic lymphocytic leukemia
B cells
after treating with EC1 (17-AAG) (;) or'EC116 (inactive structurally-related
HSP90 inhibitor)
(M). It is obviously show that ZAP70+ CLL B cells were readily killed by 17-
AAG, with a 50%
inhibitory concentration (IC50) of approximately 80 nM.
A similar experiment was performed to measure the rate at which ZAP70+ CLL B
cells
succumb. Primary isolates of CLL B cells from an individual ZAP70+ patient
were treated with
100 nM EC1 (1 7-AAG) or EC116 (inactive structurally-related HSP90 inhibitor)
for varying
times'at 37 C. Apoptotic cells were identified by a standard protocol using
the mitochondrial
vital dye DiOC6 and propidium iodide staining. The % viability is expressed as
100% -%
apoptotic cells. FIGURE 8 compares the % viability of ZAP70+ chronic
lymphocytic leukemia
B cells after treating with 100 nM of EC1 (17-AAG) (0) or EC116 (inactive
structurally-related
HSP90 inhibitor) (+). The result indicates that ZAP70+ tumor cells were
rapidly killed by 17-
AAG, with a 50% of the cells succumbing in approximately 48 hours.
Primary isolates of CCL B cells from sixteen ZAP70+ patients and eleven ZAP70-
patients were treated with 100 nM EC1 (17-AAG) for48 hours at 37 C. Apoptotic
cells were
identified by a standard protocol using the mitochondrial vital dye DiOC6 and
propidium iodide
staining. The % viability is expressed as 100% - % apoptotic cells. FIGURE 9
compares the
viability of CLL B cells from the sixteen ZAP70+ patients and the eleven ZAP70-
patients after
treatment with lOOnM ECl (17-AAG) for 48 hours. ZAP70+ CLL B cells have an
average %
viability of 45.74 +/- 3.177%, whereas ZAP70- B CLL cells have an average %
viability of 93
+/-1.701 %. The Students T-Test P-value of the difference in survival between
the two
populations was < 0.0001 which is highly statistically significant.
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V. FORMULATION AND ADMINISTRATION OF PHARMACEUTICAL COMPOSITIONS
Geldanamycin may be prepared according to U.S. Patent No. 3,595,955 using the
subculture of Streptomyces hygroscopicus that is on deposit with the U.S.
Department of
Agriculture, Northern Utilization and Research Division, Agricultural
Research, Peoria, Ill.,
USA, accession number NRRL 3602. Numerous derivatives of this compound may be
fashioned
as specified in U.S. Patent Nos. 4,261,989, 5,387,584, and 5,932,566,
according to standard
techriiques.
Those of ordinary skill in the art are familiar with formulation and
administration
techniques that can be employed in use of the invention, e.g.; as discussed in
Goodman and
Gilman's, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, current edition; Pergamon
Press;
and REMINGTON'S PHARMACEUTICAL SCIENCES (current edition.) Mack Publishing
Co.,
Easton, Pa.
The compounds utilized in the methods of the instant invention may be
administered
either alone or in combination with pharmaceutically acceptable carriers,
excipients or diluents,
in a pharmaceutical composition, according to standard pharmaceutical
practice. The
compounds can be administered orally or parenterally, including the
intraventous, intramuscular,
intraperitoneal, subcutaneous, rectal and topical routes of administration.
For example, the therapeutic or pharmaceutical compositions of the invention
can be
administered locally to the area in need of treatment. This may be achieved
by, for example, but
not limited to, local infusion during surgery, topical- application, e.g.,
cream, ointment, injection,
catheter, or implant, said implant made, e.g., out of a porous, nonporous, or
gelatinous material,
including membranes, such as sialastic membranes, or fibers. The
administration can also be by
direct injection at the site (or former site) of a tumor or neoplastic or pre-
neoplastic tissue.
.25 Still further, the therapeutic or pharmaceutical composition can be
delivered in a vesicle,
e.g., a liposome (see, for example, Langer, Science,1990, 249:1527-1533; Treat
et al.,
Liposomes in the Therapy ofInfectious Disease and Cancer, 1989, LopezBernstein
and Fidler
(eds.), Liss, N.Y:, pp. 353-365). .
The pharmaceutical compositions used in the methods of the present invention
can be
delivered in a controlled release system. In one embodiment, a pump may be
used (see, Sefton,
CRC Crit. Ref. Biomed. Eng.1987,14:201; Buchwald, et al., Surgery,1980,
88:507; Saudek et
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al.,1V. Engl. J. Med., 1989, 321:574). Additionally, a controlled release
system can be placed in
proximity of the therapeutic target. (see, Goodson, Medical Applications of
Controlled Release,
1984, Vol. 2, pp. 115-138).
The pharmaceutical compositions used in the methods of the instant invention
can
contain the active ingredient in a form suitable for.oral use, for example, as
tablets, troches,
lozenges, aqueous or oily suspensions, dispersible powders or
granules,,emulsions, hard or soft .
capsules, or syrups or elixirs. Compositions intended for oral use may be
prepared according to
any method known to the art for the manufacture of pharmaceutical compositions
and such
compositions may contain one or more agents selected from the group consisting
of sweetening
agents, flavoring agents, coloring agents and preserving agents in order to
provide
pharmaceutically elegant and palatable preparations. Tablets contain the
active ingredient in
admixture with non-toxic pharmaceutically acceptable excipients which are
suitable for the
manufacture of tablets. These excipients may be, for example, inert diluents,
such as calcium
carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and
disintegrating agents, such as microcrystalline cellulose, sodium
crosscarmellose, corn starch, or
.alginic acid; binding agents, for example starch, gelatin, polyvinyl-
pyrrolidone or acacia, and
lubricating agents, for example, magnesium stearate, stearic acid or talc. The
tablets may be
uncoated or they may be coated by known techniques to mask the taste of the
drug or delay
disintegration and absorption in the gastrointestinal tract and thereby
provide a sustained action
over a longer period. For example, a water soluble taste masking material such
as
hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay
material such as ethyl
cellulose or cellulose acetate butyrate, may be employed.
Formulations for oral use may also be presented as hard gelatin capsules
wherein the
active ingredient is mixed with an inert solid diluent, for example, calcium
carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient
is mixed with water
soluble carrier such as polyethyleneglycol or an oil medium, for example
peanut oil, liquid
paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients
suitable for
the manufacture of aqueous suspensions. Such excipients are suspending agents,
for example
30* sodium carboxymethylcellulose, methylcellulose, hydroxyprop.ylmethyl-
cellulose, sodium
alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents
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may be a naturally-occurring phosphatide, for example lecithin, or
condensation products of an
alkylene oxide with fatty acids, for example polyoxyethylene stearate, or
condensation products
of ethylene oxide with long chain aliphatic alcohols, for'example
heptadecaethylene-oxycetanol,
or condensation products of ethylene oxide with partial esters derived from
fatty acids and a
hexitol such as polyoxyethylene sorbitol monooleate, or condensation products
of ethylene oxide
with partial esters derived from fatty acids and hexitol anhydrides, for
example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one or more
preservatives, for
example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one
or more
flavoring agents, and one or more sweetening agents, such as sucrose,
saccharin or aspartame.
' Oily suspensions may be formulated by suspending the active ingredient in a
vegetable
oil, for exampie arachis bil;.olive oil, sesame oil or coconut oil, or
irimineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for example
beeswax, hard
paraffin or cetyl alcohol. Sweetening agents such as those set forth above and
flavoring agents
may be added to provide a palatable oral preparation. These compositions may
be preserved by
the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-
tocopherol.
Dispersible powders and granules suitable for preparation of an aqueous
suspension by
the addition of water provide the active ingredient in admixture with a
dispersing or wetting
agent, suspending agent and one or more preservatives. Suitable dispersing or
wetting agents
and suspending agents are exemplified by those already mentioned above.
Additional excipients,
for example sweetening, flavoring and coloring agents, may also be present.
These compositions
may be preserved by the addition of an anti-oxidant such as ascorbic acid. .
The pharmaceutical.compositions used in the methods of the instant invention
may also
be in the form of an oil-in-water emulsions. The oily phase may be a vegetable
oil, for example
olive oil or arachis oil, or a mineral. oil, for. example liquid paraffin or
mixtures of these. Suitable
emulsifying agents may be naturally-occurring phosphatides, for example soy
bean lecithin, and
esters or partial esters derived from fatty acids and hexitol anhydrides, for
example sorbitan
monooleate, and condensation products of the said partial esters with ethylene
oxide, for
example polyoxyethylene sorbitan monooleate. The emulsions may also contain
sweetening,
flavoring agents, preservatives and antioxidants.
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Syrups and elixirs may be formulated with sweetening agents, for example
glycerol,
propylene glycol, sorbitol or sucrose. Such formulations may also contain a
demulcent, a
preservative, flavoring and coloring agents and antioxidant.
The pharmaceutical compositions may be in the form of sterile injectable
aqueous
solutions. Among the acceptable vehicles and solvents that may be employed are
water,
Ringer's solution and isotonic sodium chloride soliution.
The sterile injectable preparation may also be a sterile injectable oil-in-
water
microemulsion where. the active ingredient is dissolved in the oily phase. For
example, the
active ingredient may. be first dissolved in a mixture of soybean oil and
lecithin. The oil solution
is then introduced into a water and glycerol mixture and processed to form a
microemulation.
The injectable solutions or microemulsions may be introduced into a patient's
blood-
stream by local bolus injection. Alternatively, it may be advantageous to
administer the solution
or microemulsion in such a way as to maintain a constant circulating
concentration of the instant
compound. In order to maintain such a constant concentration, a continuous
intravenous
delivery device may be utilized. An example of such a device is the Deltec
CADD-PLUSTM
model 5400 intravenous pump.
The pharmaceutical compositions may be in the form of a sterile injectable
aqueous or
oleagenous suspension for intramuscular and subcutaneous administration. This
suspension may
be formulated according to the known art using those suitable dispersing or
wetting agents and
suspending agents which have been mentioned above. The sterile injectable
preparation may.
also be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent
or solvent, for example as a solution=in 1,3-butane diol. In addition,
sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this purpose
any bland fixed
oil may be employed including synthetic mono- or diglycerides. In addition,
fatty acids such as
oleic acid find use in the preparation of injectables.
The HSP90 inhibitors used in the methods of the present invention may also be,
administered in the form of suppositories for rectal administration of the
drug. These
compositions can be prepared by mixing the inhibitors with a suitable non-
irritating, excipient
which is solid at ordinary temperatures but liquid at the rectal temperature
and will therefore
melt in the rectum to release the drug. Such materials include cocoa butter,
glycerinated gelatin,
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hydrogenated vegetable oils, mixtures of polyethylene glycols of various
molecular weights and
fatty acid esters of polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or suspensions, etc.,
containing an
HSP90 inhibitor can be used. As used herein, topical application can include
mouth washes and
gargles.
The compounds used in the methods of the present invention can be administered
in
intranasal form via topical use of suitable intranasal vehicles and delivery
devices, or via'.
transdermal routes, using those forms oftransdermal skin patches well known to
those of
ordinary skill in the art. To be adminis#ered in the form of a transdermal
delivery system, the
dosage administration will, of course, be continuous rather than interAnittent
throughout the
dosage regimen.
The methods and compounds of the instant invention may also be used in
conjunction
with'other well known therapeutic agents that are selected for their
particular usefulness against
the condition that is being treated. For example, the instant compounds may be
useful in
combination with known anti-cancer and cytotoxic agents.
Further, the instant methods and compounds may also be iiseful in combination
with
other inhibitors of parts of the signaling pathway that links cell surface
growth factor receptors to
nuclear signals initiating cellular proliferation.
The methods of the present invention may also be useful with other agents that
inhibit
angiogenesis and thereby inhibit the growth and invasiveness of tumor cells,
including, but not
limiteci to VEGF receptor inhibitors, including ribozymes and antisense
targeted to VEGF
receptors, angiostatin and endostatin.
Examples of antineoplastic agents, which can be used in combination with the
methods of
the present invention include, in general, alkylating agents, anti-
metabolites; epidophyllotoxin;
an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine;
mitoxantrone; platinum
coordination complexes; biological response modifiers -and growth inhibitors;
hormonal/anti-
hormonal therapeutic agents and haematopoietic growth factors.
Example classes of antineoplastic agents include, for example, the
anthracycline family
of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic
nucleosides, the
epothilones, discodermolide, the pteridine family of drugs, diynenes and the
podophyllotoxins.
Particularly useful members of those classes include, for example,
camiinomycin, daunorubicin,
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aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C,
porfiromycin,
5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside,
podophyllotoxin or podo-
phyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide,
melphalan,
vinblastine, vincristine, leurosidine, vindesine, leurosine, paclitaxel and
the like. Other useful
antineoplastic agents include estramustine, carboplatin, cyclophosphamide,
bleomycin,
gemcitibine, ifosamide, melphalan, hexamethyl melamine, thiotepa, cytarabin,
idatrexate,
trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT- 11, topotecan,
ara-C,
bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives,
interferons and interleukins.
When a HSP90 inhibitor used in the methods of the present invention is
administered into
a human subject, the daily dosage will normally be determined by the
prescribing physician with
the dosage generally varying according to the age, weight, and response of the
individual patient,
as well as the severity of the patient's symptoms.
In one exemplary application, a suitable amount of a HSP90 inhibitor is
administered to a'
mammal undergoing treatment for cancer,, for example, breast cancer.
Administration occurs in
an amount of each type of inhibitor of between about 0.1 mg/kg of body weight
to about
60 mg/kg of body weight per day, preferably of between 0.5 mg/kg of body
weight to about
40 mg/kg of body weight per day. A particular therapeutic dosage that
comprises the instant
composition includes from about 0.01 mg to about 1000 mg of a HSP90 inhibitor.
Preferably,
the dosage comprises from about 1 mg to about 1000 mg of a HSP90 inhibitor.
Preferably, the pharmaceutical preparation is in unit dosage form. In such
form, the
preparation is subdivided into unit doses containing appropriate quantities of
the active
component, e.g., an effective amount to achieve the desired purpose.
The quantity of active compound in a unit dose of preparation may be varied or
adjusted
from about 0.1 mg to 1000 mg, preferably from about 1 mg to 300 mg, more
preferably 10 mg to
200 mg, according to the particular application.
The actual dosage employed may be varied depending upon the requirements of
the
patient and the severity of the condition being treated. Determination of the
proper dosage for a
particular situation is within the skill of the art. Generally, treatment is
initiated with smaller
dosages which are less than the optimum dose of the compound. Thereafter, the
dosage is
increased by small amounts until the optimum effect under the circumstances is
reached. For
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convenience, the total daily dosage may be divided and administered in
portions during the day if
desired.
The amount and frequency of administration of the HSP90 inhibitors used in the
methods
of the present invention and if applicable other chemotherapeutic agents
and/or radiation therapy
will be regulated according to the judgment of the attending clinician
(physician) considering
such factors as age, condition and size of the patient as well as severity of
the disease being
treated.
The chemotherapeutic agent and/or radiation therapy can be administered
according to
therapeutic protocols well known in the art. It will be apparent to those
skilled in the art that the
administration of the chemotherapeutic agent and/or radiation therapy can be
varied depending
on the disease being treated and the known effects of the chemotherapeutic
agent and/or
radiation therapy on that disease. Also, in accordance with the knowledge of
the skilled
clinician; the therapeutic protocols (e.g., dosage amounts and times of
administration) can be.
varied in view of the observed effects of the administered therapeutic agents
(i.e., antineoplastic
agent or radiation) on the patient, and in view of the observed responses of
the disease to the
administered therapeutic agents.
Also, in general, the HSP90 inhibitor and the chemotherapeutic agent do not
have to be
administered in the same phanmaceutical composition, and may, because of
different physical
and chemical characteristics, have to be administered by different routes: For
example, the
HSP90 inhibitor may be administered orally to generate and maiiitain good
blood levels thereof,
while the chemotherapeutic agent may be administered intravenously. The
determination of the
mode of administration and the advisability of administration, where possible,
in the same
pharmaceutical composition, is well within the knowledge of the skilled
clinician. The initial
administration can be made according to established protocols known in the
art, and then, based
upon the observed effects, the dosage, modes of administration and times of
administration cari
be modified by the skilled clinician.
The particular choice of HSP90 inhibitor, and chemotherapeutic agent and/or
radiation
will depend upon the diagnosis of the attending physicians and their judgment
of the condition of
the patient and the appropriate treatment protocol.
The HSP90 inhibitor, and chemotherapeutic agent and/or radiation may be
administered
concurrently (e.g., simultaneously, essentially simultaneously or within the
same treatment
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protocol) or sequentially, depending upon the nature of the proliferative
disease, the condition of
the patient, and the actual choice of chemotherapeutic agent and/or radiation
to be administered
in conjunction (i.e., within a single treatment protocol) with the HSP90
inhibitor.
,If the HSP90 inhibitor and the chemotherapeutic agent and/or radiation are
not
administered simultaneously or essentially simultaneously, then the initial
order of
administration of the HSP90 inhibitor and the chemotherapeutic agent and/or
radiation may not
be important. Thus, the HSP90 inhibitor may be administered first followed by
the
administration of the chemotherapeutic, agent and/or radiation; or the
chemotherapeutic agent
and/or radiation may be administered first followed by the administration of
the HSP90 inhibitor.
This altemate administration may be repeated during a single treatment
protocol. The
determination of the order of administration, and the number -of repetitions
of administration of
each therapeutic agent during a treatment protocol, is well within the
knowledge of the skilled
physician after evaluation of the disease being treated and the condition of
the patient. For example, the chemotherapeutic agent and/or radiation may be
administered first, especially if it
is a cytotoxic agent, and then the treatment continued with the administration
of the HSP90
inhibitor followed, where detennined advantageous, by the administration of
the
chemotherapeutic agent and/or radiation, and so on until the treatment
protocol is complete.
Thus, in accordance with experience and knowledge, the practicing physician
can modify
each protocol'for the administration of a component (therapeutic agent-i. e.,
HSP90 inhibitor, ,
chemotherapeutic agent or radiation) of the treatment according to the
individual patient's needs,
as the treatment proceeds.
The attending clinician, in judging whether treatment is effective at the
dosage
administered, will consider the general well-being of the patient as well as
more definite signs
such as relief of disease-related symptoms, inhibition of tumor growth, actual
shrinkage of the
tumor, or inhibition of metastasis. Size of the tumor can be measured by.
standard methods such
as radiological studies, e.g., CAT or MRI scan, and successive measurements
can be used to ..
judge whether or not growth of the tumor has been retarded or even reversed.
Relief of disease-
related symptoms such as pain, and improvement in overall condition cati also
be used to help
judge effectiveness of treatment.
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VI. METHOD OF USING THE FORMULATIONS
A. Dose Range
A phase I pharmacologic study of 17-AAG in adult patients with advanced solid
tumors
determined a maximum tolerated dose (MTD) of 40 mg/mz when administered daily
by 1 -hour
infusion for 5 days every three weeks. (Wilson et al., Am. Soc. Clin. Oncol.,
abstract, "Phase I
Pharmacologic Study of 17-(Allylamino)-17-Demethoxygeldanamycin (AAG) in Adult
Patients
with Advanced Solid Tumors" 2001.) In this study, mean SD values for
terminal half-life,
clearance and steady-state volume were determined to be 2.5 0.5 hours, 41.0
13.5 L/hour,
and 86.6 34.6 L/m2, respectively. Plasma Cmax levels were determined to be
1860 660 nM
and 3170 1310 nM at 40 and 56 mg/mZ. Using these values as guidance, it is
anticipated that
the range of useful patient dosages for formufations of the present invention
will include between
about 0.40 mg/m2 and 4000 mg/mz of active ingredient, where rn2 represents
surface area.
Standard algorithms exist to convert mg/ma to mg of drug/kg patient
bodyweight.
EXAMPLES
The following examples are offered by way of illustration only, and all drugs,
components, molar ratios, concentrations, pH and steps included therein are
not intended to be
limiting of the invention unless specifically recited in the claims. Compound
preparations of
Examples 1-12 are reproduced appropriately below, from commonly owned U.S.
Provsional
Application Ser. Nos. 60/371,665 and 60/478,430, and International Application
PCT/US03/10533, entitled NOVEL ANSAMYCIN FORMULATIONS AND METHODS FOR
PRODUCING
AND USING SAME, filed Apri14, 2003, and International Application PCT/US
03/1053, entitled
DRUG FORMULATIONS HAVING LONG AND MBDIUM CHAIN TRIGLYCERIDES, filed October 4,
2003, and to which this application claims priority.
Example 1: Preparation of 17-AAG
To 45.0 g (80.4 mmol) of geldanamycin, in 1.45 L of dry THF in a dry 2 L flask
was
added drop-wise over 30 minutes 36.0 mL (470 rnmol) of allyl amine in 50 mL of
dry THF. The
reaction mixture was stirred at room temperature under nitrogen for 4 hr at
which time TLC
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analysis indicated the reaction was complete [(GDM: bright yellow: Rf=0.40;
(5% MeOH-95%
CHC13); 17-AAG: purple: Rf--0.42 (5% MeOH-95% CHCl3)]. The solvent was removed
by rotary evaporation and the crude material was slurried in 420 mL of
H20:EtOH (90:10) at 25 C,
filtered and dried at 45 C for 8 hr to give 40.9 g (66.4 mmol) of 17-AAG as
purple crystals
(82.6 % yield, > 98% pure by HPLC monitored at 254 nm). MP 206-212 C. 'H NMR
and
HPLC are consistent with the desired product.
Example 2: Preparation of a Low Melt Form of 17-AAG
The crude 17-AAG from Example 1 is dissolved in. 800 mL of 2-propyl alcohol
(isopropanol) at 80 C and then cooled to room temperature. Filtration
followed by drying at
45 C for 8 hr gives 44.6 g (72.36 mmol) of 17-AAG as purple crystals (90%
yield, > 99% pure
by HPLC monitored at 254 nm). MP = 147-153 C. "H NMR and HPLC are consistent
with the
desired product.
Example 3: Solvant Stability of a Low Melt Form of 17-AAG
The 17-AAG product from Example 2 in 400 mL of H20:EtOH (90:10) at 25 C,
filter
and dry at 45 C for 8 hr to give 42.4 g (68.6 mmol) of 17-AAG as purple
crystals (95 % yield, >
99% pure by HPLC monitored at 254 nm). MP = 147-175 C. 'H NMR and HPLC are
consistent with the desired product.
Example 4: Preparation of Compound 237: A dimer
3,3-diamino-dipropylamine (1.32 g, 9.1 mmol) was added dropwise to a solution
of
geldanamycin (10 g, 17.83 mmol)'in DMSO (200 mL) in a flame-dried flask under
NZ and stirred
at room temperature. The reaction mixture was diluted with water after 12
hours. A precipitate
was formed and filtered to give the crude product. The crude product was
chromatographed by
silica chromatography (5% CH3OH/CH2Cl2) to afford the desired dimer as a
purple solid. The
pure purple product was.obtained after flash chromatography (silica gel);
yield: 93%; mp 165 C;
'H NMR (CDC13) 0.97 (d, J= 6.6 Hz, 6H, 2CH3),1.0 (d, J = 6.6 Hz, 6H, 2CH3),
1.72 (m, 411,
2 CH2),1.78 (m, 4H, 2CH2), 1.80 (s, 6H, 2CH3), 1.85 (m, 2H, 2CH), 2.0 (s, 6H,
2CH3), 2.4 (dd, J
11 Hz, 2H, 2CH), 2.67 (d, J= 15 Hz; 2H, 2CH), 2.63 (t, J= 10 HZ, 211, 2CH),
2.78 (t, J= 6.5
Hz, 4H, 2CH2), 3.26 (s, 6H, 20CH3), 3.38 (s, 6H, 20CH3), 3.40 (m, 2H, 2CH),
3:60 (m, 4H,
2CH2), 3.75 (m, 2H, 2CH), 4.60 (d, J= 10 Hz, 2H, 2CH), 4.65 (Bs, 2H, 20H),
4.90 (Bs, 4H,
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2NH2), 5.19 (s, 2H, 2CH), 5.83 (t, J = 15 Hz, 2H, 2CH=), 5.89 (d, J 10 Hz, 2H,
2CH=), 6.58
(t, J=15 Hz, 2H, 2CH=), 6.94 (d, J=10 Hz, 211, 2CH=), 7.17 (m, 2H, 2NH ), 7.24
(s, 211,
2CH=), 9.20 (s, 2H, 2N-H); MS (m/z) 1189 (M H).
The corresponding HCl salt was prepared by the following method: an HCl
solution in
EtOH (5 ml, 0.12 3N) was added to a solution of compound #237 (1 g as prepared
above) in
THF (15 ml) and EtOH (50 ml) at room temperature. The reaction mixture was
stirred for
min. The salt was precipitated, filtered and washed with a large amount of
EtOH and dried in
vacuo. Alternatively, a "mesylate" salt can be formed using methanesulfonic
acid instead of
HC1.
10 Example 5: Preparation of Compound 914
To geldanamycin (500 mg, 0.89 mmol) in 10 mL of dioxane was added selenium
(IV)
dioxide (198 mg, 1.78 mmo 1) at room temperature. The reaction mixture was
heated to 100 C
and stirred for 3 hours. After cooling to room temperature, the solution was
diluted with ethyl
acetate, washed with water and brine, dried over magnesium sulfate, filtered
and evaporated in
vacuo.The fmal pure yellow product was obtained after column chromatography
(silica gel);
yield: 75%; 'H NMR (CDC13) S 0.97(d, J=7.OHz, 3H, CH3),1.01(d, J=7.OHz, 3H,
CH3),
1.75(m, 3H, CH2+CH), 2.04(s, 311, CH3), 2.41(d, J=9.9Hz, 1H, CHa), 2.53(t,
J=9.9Hz,1H, CH2),
2.95(m,1H, CH), 3.30(m, 2H, CH+OH), 3.34(s, 6H, 2CH3), 3.55(m, 1H, CH),
4.09(m, 1H, CHz),
4.15(s, 3H, CH3), 4.25(m,1H, CH2), 4.41(d, J=9.8Hz, 1H, CH), 4.80(bs, 2H,
CONHa), 5.32(s,
1H, CH), 5.88(t, J=10.4Hz, 1H, CH=), 6.04(d, J=9.7Hz,1H, CH=), 6.65(t,
J=11.5Hz,1H, CH=),
6.95(d, J=11.5Hz,1H, CH=), 7.32(s,1H, CH-Ar), 8.69(s,'1H, NH); MS (m/z) 575.6
(M-1).
Example 6: Preparation of Compound 967
To compound #914 (50 mg, 0.05 mmol) in 3mL of THF was added allylamine (3.5
mg,
0.06 mmol). The reaction mixture was stiured at room temperature for 24 hours.
The solvent
was removed by rotary evaporation. The pure purple product was obtained after
column
chromatography (silica gel); yield: 90%; 'H NMR (CDC13) 50.89(d, J=6.6Hz, 3H,
CH3),
1.03 (d, J=6.9Hz, 3H, CH3),1.78(m,1H, CH), 1.82(m, 2H, CHZ), 2.04 (s, 3H,
CH3),
2.37(dd, J=13.7Hz,1H, CHa), 2.65(d, J=13.7Hz,1H, CHz), 2.90(m; 1H, CH),
3.33(s, 3H, CH3),
3.34(s, 3H, CH3), 3.45(m, 2H, CH+OH), 3.58(m, 1H, CH), 4.14(m, 3H, CH2+CH2),
4.16(m, 1H, CH2), 4.42(s,1H, OH), 4.43(d, J=10Hz,1H, CH), 4.75(bs, 211,
CONH2),
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5.33(m, 2H, CH2=), 5.35(s, I H, CH), 5.91(m, 2H, CH=+CH=), 6.09(d, J=9.9Hz,
1H, CH=),
6.46(t, J=5.8Hz, 1H, NH), 6.66(t, J=11.6Hz, 1H,-CH=), 6.97(d, J=11.6Hz, 1H,
CH=),
7.30(s, 1H, CH), 9.15(s, 111, NH).
Example 7: Preparation of Compound 956
Compound #956 was prepared by the same method described for compound #967
except
that azetidine was used instead of allylamine. The final pure purple product
was obtained after
column chromatography (silica gel); yield: 89%; IH NMR (CDCI 3) S 0.99 (d, J =
6.8.Hz, 3H,
CH3), 1.04 (d, J = 6.8 Hz, 3H, CH3),1.77 (m, 1H, CH), 1.80 (m, 2H, CH2), 2.06
(s, 3H, CH3),
2.26 (m, 1H, CHa), 2.50(m, 2H, CHZ), 2.67 (d, 1H, CH2), 2.90 (m, 1H, CH), 3.34
(s, 3H, CH3),
3.36 (s, 3H, CH3), 3.48 (m, 2H, OH+CH), 3.60 (t, J= 6.8Hz, 1H, CH), 4.11 (dd,
J=12Hz,
J=4.5Hz, 1H, CH2), 4.30 (dd, J=12Hz, J=4.5Hz,1H, CHa),'4.45 (d, J= Y0.0Hz, 1H,
CH),
4.72 (m, 5H, 2CHa+OH), 4.78 (bs, 211, NH2), 5.37 (s, 1H, CH), 5.89 (t,
J=10.5Hz, 1 H, CH=),
6.10 (d, J=10 Hz, 1 H, CH=), 6.66 (t, J=12Hz, 1 H, CH=), 7.00 (d, J=12Hz,1H,
CH=), .
7:17 (s, 1H, CH=), 9.20 (s, 1H, CONH); MS(m/z) 602 (M+1).
Example 8: Preparation of Compound 529
A solution of 17-aminogeldanamycin (1 mmol) in EtOAc was treated with Na2S204
(0.1 M, 300 ml) at RT. After 2 h, the aqueous layer was extracted twice with
EtOAc and the
combined organic layers were dried over Na2SO4, concentrated under reduce
pressure to give
18,21-dihydro-17-aminogeldanamycin as a yellow solid. This solid was dissolved
in anhydrous
THF and transferred via cannula to a mixture of picolinoyl chloride (1.1 mmol)
and MS4A
(1.2 g). Two hours later, EtN(i-Pr)2 (2.5 mmol) was further added to the
reaction mixture. After
overnight stirring, the reaction mixture was filtered and concentrated under
reduce pressure.
Water was then added to the residue, which was extracted with EtOAc three
times; the combined
organic layers were dried over NaaSO4 and concentrated under reduce pressure
to give the crude
product which was purified by flash chromatography to give 17-picolinoyl-
aminogeldanamycin,
Compound 529, as a yellow solid. Rf = 0.52 in 80:15:5 CHZCIZ: EtOAc: MeOH.
Mp = 195-197 C. 1H NMR (CDC13) S 0.91 (d, 3H), 0.96 (d, 3H), 1.71-1.73 (in,
2H), 1.75-1.79
(m, 4H), 2.04 (s, 3H), 2.70-2.72 (m, 2H), 2.74-2.80 (m, 1H), 3.33-3.35 (m,
7H),
3.46-3.49 (m, 1H), 4.33 (d, 111), 5.18 (s, 1H), 5.77 (d, 1H), 5.91 (t, 1H),
6.57 (t, 1H),
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6.94 (d, 1H), 7.51-7.56 (m, 2H), 7.91 (dt, 1H), 8.23 (d, IH), 8.69-8.70
(m,1H), 8.75(s, 1H),
10.51 (s, 1 H).
Example 9: Preparation of Compound 1046
Compound #1046 was prepared according to the procedure described for compound
#529
using 4-chloromethyl-benzoyl chloride instead of picolinoyl chloride. (3.1 g,
81 %). - Rf = 0.45 in
80:15:5 CH2Clz: EtOAc: MeOH. 1H NMR CDC13 S 0.89 (d, 3H), 0.93 (d, 3H), 1.70
(br s, 2H),
1.79 (br s, 41-1), 2.04 (s, 311), 2.52-2.58 (m, 2H), 2.62-2.63 (m, IH), 2.76-
2.79 (m, I H),
3.33 (br s, 7H), 3.43-3.45 (m, 1H), 4.33 (d, 1H), 4.64 (s, 2H), 5.17 (s, 1H),
5.76 (d, 1H),
5.92 (t,1 H), 6.57 (t,1 H), 6.94 (d, 1 H), 7.49 (s,1 H), 7.5 5(d, 211), 7.91
(d, 2H), 8.46 (s, 1 H),
8.77 (s, 1 H).
Example 10: Preparation of Compound 1059
To a solution of compound #1046 (0.14 g, 0.2 mmol) in THF (5 ml) were added
sodium
iodide (30 mg, 0.2 mmol) and morpholine (35 L, 0.4 mmol). The resulting
mixture was heated
at reflux for 10h whereupon it was cooled to room temperature, concentrated
under reduce
pressure and the residue was redissolved in EtOAc (30 ml),'washed with water
(10 ml), dried
with Na2SO4 and concentrated again. The residue was then recrystallized in
EtOH (10 ml) to
give the compound 1059 as a yellow solid (100 mg, 66%). Rf-- 0.10 in 80:15:5
CH2C12: EtOAc:
MeOH.1H NMR CDC13 8 0.93 (s, 3H), 0.95 (d, 3H), 1.70 (br s, 2H),1.78 (br s,
4H),
2.03 (s, 3H), 2.48 (br s, 4H), 2.55-2.62 (m, 3H), 2.74-2.79 (m, 1H), 3.32 (br
s, 7H), 3.45 (m, IH),
159- (s, 2H), 3.72-3.74 (m, 4H); 4.32 (d, 1H), 5;15 (s, 1H), 5.76 (d, 1H),
5.91 (t, 1H),
6.56 (t, 1H), 6.94 (d, 1H), 7.48 (s,1H),.7.50 (d, 2H), 7.87 (d, 211), 8.47
(s,1H), 8.77 (s,1H).
Example 11: Preparation of Compound 1236
Compound #1236 was prepared according to the procedure described for compound
#1059 using benzylethyl amine instead of morpholine. Rf = 0.43 in 80:15:5
CH2Cla: EtOAc:
MeOH. 1H NMR CDC13 S 0.925 (s, 3H), 0.95 (d, 3H), 1.09 (t) 3H), 1.70 (br..s,
2H),
1.79 (br s, 4H), 2.04 (s, 3H), 2.50-2.62 (m, 5H), 2.75-2.79 (m, IH), 3.32 (br
s, 7H), 3.46 (m,.1H),
3.59 (s, 2H), 3.63 (s, 2H), 4.33 (d, 1H), 5.16 (s,1H), 5.78 (d, 1H), 5.91
(t,1H), 6.57 (t, 1H),
6.94 (d, 1H), 7.25-7.27 (m, 11-1), 7.32-7.38 (m, 4H), 7.48 (s, 1H), 7.53 (d,
2H), 7.85 (d, 2H),
8.47 (s, 1 H), 8.77 (s, I H).
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Example 12: Preparation of Compound 563: 17-(benzoyl)-'
aminogeldanamycin.
A solution of 17-aminogeldanamycin (1 mmol) in EtOAc was treated withNa2S204
(0.1 M, 300 mL) at RT. After 2 h, the aqueous layer was extracted twice with
EtOAc and the
combined organic layers were dried over Na2SO4, concentrated under reduce
pressure to give
18,21-dihydro-17aminogeldanamycin as a yellow solid. This solid was dissolved
in anhydrous
THF and transferred via cannula to a mixture of benzoyl chloride (1.1 mmol)
and MS4A'(1.2 g).
Two hours later, EtN(i-Pr)2 (2.5 mmol) was further added to the reaction
mixture. After
ovemight stirring, the reaction mixture was filtered and concentrated under
reduce pressure.
Water was then added to the residue which was extracted with EtOAc three
times, the combined
organic layers were dried over NaaSO4 and concentrated under reduce pressure
to give the crude
product which was purified by flash chromatography to give 17-(benzoyl)-
aminogeldanamycin.
Rf = 0.50 in 80:15:5 CH2C12: EtOAc: MeOH. Mp = 218-220 C. 1H NMR (CDC13) 0.94
(t, 6H),
1.70 (br s, 2H), 1.79 (br s, 4H), 2.03 (s, 3H), 2.56 (dd, 1H), 2.64 (dd, I H),
2.76-2.79 (m, I H),
3.33 (br s, 7H), 3.44-3.46 (m, 1H), 4.325 (d, I H), 5.16 (s,1H), 5.77 (d, 1H),
5.91 (t, 1H),
6.57 (t, 1H), 6.94 (d, 1H), 7.48 (s, 1H), 7.52 (t, 2H), 7.62 (t,1H), 7.91 (d,
2H), 8.47 (s, 1H),
8.77 (s, IH).
Example 13: Preparation of cell lysates.
Cells for the study were lysed in lysis buffer (20 mM HEPES, pH 7.3, 1 mM
EDTA,
5 mM MgC12, 100 mM KCl) by manual douncing in a Potter-Elvejem homogenizer.
Example 14: HSP90 lysate binding assays.
Normal B cell, normal T cell, ZAP70+ CLL B cells and ZAP70- CLL B cells were
lysed
in lysis buffer as described in Example 13. The lysates were incubated with or
without 17-AAG
for 30 mins at 4 C, and then incubated with biotin-GM linked to BioMagTM
streptavidin
magnetic beads (Qiagen) for 1 hr at, 4 C. Tubes were placed on a magnetic
rack, and the
unbound supematant removed. The magnetic beads were washed three times in
lysis buffer and
boiled for 5 min at 95 C inSDS-PAGE,sample buffer. Samples were analyzed on
SDS protein
gels, and Western blots done using an HSP90 antibody (StressGen, SPA-830).
Bands in the
Western Blots were quantitated using the Bio-rad Fluor-S Multilmager, and the
% inhibition of
binding of HSP90 to the biotin-GM was calculated. The IC5o reported is the
.concentration of 17-
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AAG needed to cause half-maximal inhibition of binding. The results of
competitive binding is
showed in Figures 1-3.
Example 15: Study to assess the association of HSP90 with client protein
MCF-7 breast carcinoma cells, primary isolates of ZAP70+ and ZAP70- B-cell
chronic
lymphocytic leukemia (B-CLL) cells and normal T and B cells were lysed as
described in
Example 13 and co-immunoprecipitation experimerits were performed as described
in Kamal et
al. Nature, 2003 425: 407-410. Protein-A Sepharose beads (Zymed) were
pre=blocked with 5%.
BSA. The cell lysates were pre-cleared by incubating with 50 L of protein-A
Sepharose beads
(50% slurry). To 100 L of the pre-cleared cell lysate,'either no antibody or
antibodies to
HSP90, p23 and Hop were added, and incubated by rotating for 1 h at 4 C. 50 L
of pre-cleared
beads (50% slurry) was then added and incubated by rotating for 1 h at 4 C.
Bound beads were
briefly centrifuged at 3,000g and unbound samples collected. Beads were washed
thrice in lysis
buffer and once with 50 mM Tris, pH 6.8, and then SDS-sample buffer added for
5 min at 95 C.
Bound and unbound samples were analysed by SDS-PAGE and western blots using
indicated
antibodies. The result of the co-immunoprecipitation study is shown in FIG. 4.
Example 16. Study to demonstrate inhibition of ZAP70 expression by
selected HSP90 inhibitors.
Primary isolates of ZAP70+ chronic lymphocytic leukemia B-cells from an
individual
ZAP70+ patient were treated with EC 1 (1 7-AAG), EC 116 (an inactive
structurally-related
HSP90 inhibitor) or EC82 or EC86 (two other known HSP90 inhibitors) for 24
hours at 37 C.
Levels of ZAP70 protein expression were measured by indirect
immunofluorescence of
permeabilized cells with specific anti-ZAP70 antibodies and FACS analysis.
Result of the study
is shown in FIG. 5. All three active HSP90 inhibitors dose-dependently induced
degradation of
ZAP70, confirming that ZAP70 is an HSP90-dependent client protein, as was
indicated by the
physical association demonstrated in the co-immunoprecipitation experiments
(FIG. 4). The fact
that three structurally-unrelated HSP90 inhibitors produced the same effect
strongly implicates
HSP90. as an essential protein for the stability of ZAP70 in CLL B-cells.
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Example 17. Study to determine downstream effect of inhibiting HSP90 on
blood cells of ZA.P70+ CCL B-cell patient.
Primary isolates of white blood cells from an individual ZAP70+ chronic
lymphocytic
leukemia B-cell patient were left untreated or treated with 300 nM 17-AAG for
24 hours at
37 C. The samples were than prepared for flow cytometry analysis by a method
described in
Rassebti et al. supra. The cells were first stained with CD19-specific and CD3-
specific
monoclonal antibodies conjugated with allophycocyanin and phycoerythrin,
respectively
(Pharmingen), and later stained with a monoclonal antibody specific for ZAP70
that has been
conjugated to Alexa-488 dye (Bectori Dickinson). CD3 is a specific marker of T
cells. Levels of
ZAP70 protein expression were measured by flow cytometry (FACSCalibur, BD
Biosciences)
and Flow-Jo sorftware, versiori 2.7.4 (Tree Star). The result is documented in
FIG. 6, the left
panel shows the ZAP70 expression in untreated cells, and the right panel shows
the ZAP70
expression of the untreated cells.
In the untreated cells (left panel), approximately 5% of the cells were normal
T-cells
(CD3+, ZAP70+, upper right quadrant) and -85% of the cells were ZAP70+ CLL B-
cells (CD3-,
ZAP70+, lower right quadrant). 17-AAG induced degradation of ZAP70 in the CLL
B-cells (%
positive cells 85% 4 34%), but not in the normal T-cells (% positive cells
4.5% 4 4.2%), as
predicted from the physical association demonstrated in CLL B-cells, but not
in the normal T-
cells in the co-immunoprecipitatioii experiments (FIG. 4).
Example 18. The concentration dependent effect of inhibiting HSP90 on
ZAP70+ CCL B cell viability.
Primary isolates of ZAP70+ chronic lymphocytic leukemia B-cells from an
individual
patient were treated with increasing concentration ofEC1 (17-AAG) or EC 116
(inactive
structurally-related HSP90 inhibitor) for 48 hours at 37 C. Apoptotic cells
were identified by a
standard protocol using the mitochondrial vital dye DiOC6 and propidium iodide
-staining.
Results were plotted in FIG. 7 of the % viability vs. concentration of the
inhibitor in nM. The %
viability is expressed as 100% - % apoptotic cells. ZAP70+ tumor cells were
readily killed by
17-AAG, with a 50% inhibitory concentration (IC50) of approximately 80nM.
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Example 19. The time dependent effect of inhibiting HSP90 on ZAP70+
CCL B cell viability.
Primary isolates of B-cell' chronic lymphocytic leukemia cells from an
individual
ZAP70 patient were treated with 100 nM EC 1 (1 7-AAG) or EC 116 (inactive
structurally-
related HSP90 inhibitor) for varying times at 37 C. Apoptotic cells were
identified by a
standard protocol using the mitochondrial vital dye DiOC6 arid propidium
iodide staining.
Results of the study were plotted iri FIG. 8 of the % viability vs. treatment
time in hours. The %
-viability is expressed as 100% - % apoptotic cells. ZAP70+ tumor cells were
rapidly killed by
17-AAG, with a 50% of the cells succumbing in approximately 48 hours:
Example 20. Downstream effect of inhibiting HSP90 in CLL B cells.
Primary isolates of chronic lymphocytic leukemia B-cells (CLL'B cells) from
sixteen
ZAP70+ patients and eleven ZAP70- patients were treated with 100 nM of 17-AAG
for 48 hours
at 37 C. Apoptotic cells were identified by a standard protocol using the
mitochondrial vital dye
DiOC6 and propidium iodide staining. Results of the study were plotted in FIG.
9. The %
viability is expressed as 100% - % apoptotic cells. ZAP70+ tumor cells were
readily killed by
17-AAG, with an average % survival of 45.74 +/- 3.177%, whereas ZAP70- cells
were
unaffected by the drug under the same conditions - survival in these cells was
93 +/-1.701%.
The Students T-Test of the difference in survival has a P-value of < 0.0001,
which is highly
statistically significant.
* * *
The foregoing examples are not intended to be limiting of and are merely
representative
of various embodiments of the invention. It will be readily apparent to one
skilled in the art that
varying substitutions and modifications may be made to the inv.ention without
departing from the
scope and spirit of the invention. Thus, such additional. embodiments are
within the scope of the
irivention'and the following claims.
Antibodies, polyclonal or monoclonal, can be purchased from a variety of
commercial
suppliers, or may be manufactured using well-known methods, e.g., as described
in Harlow et
al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory
Press, Cold
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CA 02584266 2007-04-16
WO 2006/050457 PCT/US2005/039816
Spring Harbor, N:Y. (1988). The reagents described herein are either
commercially available,
e.g., from Sigma-Aldrich, or else readily producible without undue
experimentation using
routine procedures known to those of ordinary skill in the art and/or
described in.publications
herein incorporated by reference.
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